Methods of treating cholesterolemia

ABSTRACT

THE METHODS OF THIS INVENTION ARE USEFUL FOR THE TREATMENT OF CHOLESTEROLEMINA IN MAMMALS. THE METHODS INVOLVE ADMINISTERING TO MAMMALS CERTAIN FLUORINATED DERIVATIVES OF ESTERS OF PHENOXY ISOBUTYRIC ACID.

Claims priority, application France, Sept. 26, 1967, 122,380; Nov. 22, 1967, 133,439 Int. Cl. A61k 27/00 U.S. Cl. 424-311 6 Claims ABSTRACT OF THE DISCLOSURE The methods of this invention are useful for the treatment of cholesterolemia in mammals. The methods involve administering to mammals certain fluorinated derivatives of esters of phenoxy isobutyric acid.

This application is a continuation-in-part of the pending U.S. patent application, Ser. No. 124,957, filed Mar. 16, 1971, now abandoned, which in turn is a continuation-inpart of U.S. patent application Ser. No. 760,973 filed Sept. 19, 1968, now U.S. Pat. No. 3,632,629.

This invention relates to methods of treating cholesterolemia and biliary lithiasis in mammals. The methods comprise administering to mammals, orally or parenterally such as subcutaneously or intramuscularly, fluorinated derivatives of the esters of phenoxy isobutyric acid. The daily dosage is within the range of 10 to 360 mg./kg. of body weight of the mammal treated, and preferably for man Within the range of 140 to 280 mg./kg. of body 'weight, depending upon the severity of the case.

The fluorinated derivatives of the esters of phenoxy isobutyric acid employed in the methods of this invention have the formula:

CH3 CH3 in which R is an alkyl radical of 1 to carbon atoms either straight or branch chained; X is trifluoromethyl, trifluoromethylthio, trifiuoromethylsulfinyl or trifiuoromethylsulfonyl.

The process of preparation of the fluorinated derivatives of the esters of the phenoxy isobutyric acid employed in the methods of this invention consists in starting with a substituted phenol in combination with a radical such as trifluoromethyl, trifluoromethylthio, trifiuoromethlsulfinyl, trifiuoromethylsulfonyl; treating it with chloroform and acetone in the presence of potash so as to obtain the corresponding phenoxy isobutyric acid. This is then esterified by an alcohol of low molecular weight such as methanol, ethanol, propanol, isopropanol, etc. in the presence of a catalyzing agent. The ethyl esters are particularly eflective because of their high cholesterol control properties.

Typical examples of the derivatives of esters of phenoxy United States Patent Oifice 3,790,681 Patented Feb. 5, 1974 isobutyric acid utilized in the methods of this invention are as follows:

EXAMPLE I Ethyl-p-trifluoromethylphenoxyisobutyrate Stage A, p-nitrotribromotoluene.-Into a 1 liter threenecked flask equipped with a mechanical agitator, an ascendant refrigerant and a dropping funnel, there are introduced 137 g. (1 mole) of p-nitrotoluene. The bromine is poured slowly into the flask heated to 195-200 C. When the introduction of the bromine is completed, the mixture is allowed to return to room temperature and the product is extracted with petroleum ether. The product is then treated with a solution of sodium hypobromide for 48 hours. When the reaction is completed, the product is dried, washed with water and recovered with methanol. 112 g. of p-nitrotribromotoluene are obtained. (Yield: 30% M.P. p. 86 0.)

Stage B, p-trifluoromethylnitrobenzene.Into a 1 liter flask equipped with a small distillation column joined to a descendant refrigerant, 112 g. (0.3 mole) of p-nitrotribro motoluene and 70 g. of pulverized antimony trifluoride are introduced. The flask is heated. When the highly exothermic reaction is completed, the product is distilled and is then extracted with ethyl ether. 40 g. of p-trifluoromethylnitrobenzene are obtained. (Yield: 70% MP. 40 C.)

Stage C, p-trifiuoromethylaniline.-Into a 1 liter threenecked flask equipped with a mechanical agitator, a thermometer and a dropping funnel are introduced 40 g. of p trifluoromethylnitrobenzene (0.2 mole) and 120 ml. of absolute ethanol. There is then poured in under agitation a solution of 240 g. of stannous chloride in 200 ml. of hydrochloric acid (d:1.19). When the reduction is completed, the mixture is neutralized with soda lye (360 B) and recovered several times with ether, then washed in 'water, dried with sodium anhydrous sulfate, and then distilled. 32 g. of p-trifluoromethylaniline are obtained. (Yield: about 100% M.P.: 32 C.)

Stage D, p-trifluoromethylphenol.Into a 1 liter threenecked flask equipped with a mechanical agitator, a thermometer and a dropping funnel there are introduced 180 ml. of water and then 27 ml. of sulfuric acid (d: 1.84). With the temperature maintained at about 20 C., 32 g. (0.2 mole) of p-trifluoromethylaniline are poured in slowly, and then drop by drop, a solution of 15 g. of sodium nitrite in 70 ml. of water. The mixture is maintained under agitation at room temperature for about one hour. The product formed is steam distilled and then recovered several times in ether. The ether is dried and distilled. 27.5 g. of p-trifluoromethylphenol are obtained. (Yield:

Stage E, p-trifluoromethylphenoxyisobutyric acid.- Into a 1 liter three-necked flask equipped with a mechanical agitator, an ascendant refrigerant and a dropping funnel there are introduced 27.5 g. (0.17 mole) of p-trifluoromethylphenol, 105 g. of acetone and 37 g. of pellets of soda. The mixture is refluxed and 25 g. of chloroform are poured slowly through the dropping funnel. When the reaction is completed, the acetone is removed under vacuum and the residue recovered in water and the pH is raised to 1 with concentrated hydrochloric acid. The product obtained is extracted in chloroform. 21 g. of p-triflu0 romethylphenoxyisobutyric acid are obtained. (Yield: 50% M.P.: C.).

Stage F, ethyl p-trifiuoromethylphenoxyisobutyrate.-- Into a 500 ml. flask equipped with an ascendant refrigerant there are introduced 21 g. (0.085 mole) of p-trifluoromethylphenoxyisobutyric acid and a mixture of ml. of absolute ethanol and 4 g. of concentrated sulfuric acid of 66 B. The mixture is refluxed for 5 hours. It is extracted in chloroform, the solvent removed and the residue distilled 13.8 g. of ethyl p-trifluoromethylphe- 3 noxyisobutyrate are obtained. (Yield: 60% boiling at 2 to 3 mm.: 9194 C.)

Gther examples of compounds useful in the practice of the methods of treatment of this invention are the corresponding methyl or isobutyl p-trifluoromethylphenoxyisobutyric acid. Such corresponding compounds are produced by employing instead of 100 ml. of ethanol, 70 ml. of anhydrous methanol or 160 ml. of isobutyl alcohol in Stage F.

Each of the active products of this invention may be incorporated in a tablet, capsule, ampule or other pharmaceutical form for administration to a mammal for the treatment of cholesterolemia or biliary lithiasis. For example, the' active product, such as ethyl-p-trifluoromeththylphenoxy isobutyrate, may be combined with pharmaceutical carriers such as lactose, starch and a small amount of magnesium stearate when incorporated in a tablet. Any one of the other active products described in Examples II, III and IV may likewise be combined with suitable pharmaceutical carriers in a tablet or other dosage form for administration to the mammal being treated.

EXAMPLE II Ethyl-p-trifiuoromethylthiophenoxyisobutyrate Stage A, p-nitrophenylmethylsulphide.-Into a 5 liter flask equipped with a mechanical agitator, an ascendant refrigerant and a dropping funnel there are introduced 315 g. (2 moles) of p-chloronitrobenzene and 1500 ml. of 95% ethyl alcohol. The mixture is refluxed with agitation. As the reflux is continued, there is added in a thin stream a solution of 350 g. of sodium sulfide-9 H 50 g. of sulphur and 2000 ml. of 95% alcohol.

Reflux is continued for /2 hour and then a solution of soda pellets in 150 m1. of water and 500 ml. of 95% alcohol is poured into the reaction mixture.

The reaction product is poured into a mixture of ice and water. The impurities are eliminated by filtration. The filtrate is acidified with hydrochloric acid (d:l:15). The clear yellow precipitate obtained is dried and washed with water. It is then dissolved in 200 ml. of soda lye (36 B) and 2000 ml. of water and poured into a liter flask equipped with a mechanical agitator, an ascendant refrigerant and a dropping funnel. Then under agitation, 380 g. of dimethyl sulfate are added. The mixture is refluxed for 1 hour, being assured that the medium is alkaline. The mixture is then allowed to return to room. temperature. The product crystallizes, is dried, washed with water, and dried at 50 C. After recrystallization in methanol, 16 g. of p-nitrophenylmethylsulphide are obtained. (Yield: 50%, M.P.: 71-72 C.)

Stage B, p-nitrophenyltrichloromethylsulphide.Into a 1 liter three-necked flask 169 g. (1 mole) of p-nitrophenylmethylsulphide are introduced and dissolved by heating in 620 ml. of chloroform. A part of the chloroform is then distilled and a stream of chlorine passed into the flask, the reaction temperature being maintained between and 25 C. The reaction being photo sensitive, it is catalyzed by a luminous flux. The reaction is completed in 4 hours.

The chloroform is distilled and the product recrystallized in acetone. 218v g. of p-nitrophenyltrichloromethylsulfide are obtained. (Yield: 80%, M.P. 94 C.)

.Stage C, p-nitrophenyltrifluoromethylsulphide.Into a i 1 liter flask equipped with a small distillation column and a descendant refrigerant, are introduced 218 g. (0.8 mole) 'of p nitrophenyltrichloromethylsulphide and 218 g. of

pulverized antimony trifluoride. The mixture of the two compositions is brought rapidly to the boiling point. It is then distilled under vacuum. The product obtained is recoveredin 900 ml. of sulfuric ether and 90 ml. of hydrochloric acid (d:1.19) diluted V2. The etherified layer is decanted and recovered several times by washing in /z hydrochloric acid. The ether is dried in anhydrous sodium sulphate and then distilled. The product is then distilled.

112.4 g. of p-nitrophenyltrifluoromethylsulfide are ob- Stage D, 1p-aminophenyltrifluoromethylsulfider lnto a 6 liter three-necked flask equipped with amechanical agitator, a thermometer and a dropping funnel, are introduced 112.4 g. (0.5 mole) of p-nitrophenyltrifiuoromethylsulfide and 400 ml. of 95% ethyl alcohol. Reduction is effected with a solution of 650 g. of stannous chloride in 490 m1. of hydrochloric acid (d:l.l9). After neutralization in soda, the product is extracted several times in ether, which is washed in water, dried in anhydrous sodium sulfate and then distilled. The product is then distilled. g. of p-aminophenyltrifluoromethylsulphide are obtained. (Yield: 83%, boiling at 10 mm.: 98-99 C.)

Stage E, p-hydroxyphenyltrifluoromethylwlphide-Into a 2 liter three-necked flask equipped with a mechanical agitator, a thermometer and a dropping funnel, there are introduced 450 ml. of water and then 100 ml. of pure sulfuric acid (d: 1.84). With the temperature being maintained at 20 C., 80 g. of p-aminophenyltrifiuoromethylsulphide are poured in, in a thin stream, and then, drop by drop, a solution of 40 g. of sodium nitrite in 180 ml. of water. The mixture is maintained under agitation at room temperature for about 1 hour. The product formed is drawn 005 by steam and the product is recovered several times in ether. The ether is dried and distilled, 68.5 g. of p-hydroxyphenyltrifluoromethylsulphide are obtained. (Yield: 85%, M.P.: 52 C., boiling at 10 mm.: -100" C.)

Stage F, p-trifluoromethylthiophenoxyisobutyric acid- Into a 1 liter three-necked flask, equipped with a mechanical agitator, a reflux refrigerant and a dropping funnel, there are introduced 260 g. of acetone, 68.5 .g. of p-trifluoromethylthiophenol and g. of soda pellets. The mixture is refluxed and 70 g. of chloroform are poured slowly into the dropping funnel. When the reaction is complete, the acetone is removed under vacuum and the residue recovered in water, then brought to a pH of 1 with concentrated hydrochloric acid. The product obtained is extracted with chloroform. 69 g. of p-trifluoromethylthiophenoxyisobutyric acid are obtained. (Yield: 70%, M.P.: 118 C.)

Stage G, ethyl p-trifluoromethylthiophenoxyisobutyrate.Into a 500 ml. flask equipped with an ascendant refrigerant, are introduced 69 g. of p-trifluoromethylthiophenoxyisobutyric acid and a mixture of 280 ml. of ab solute ethyl alcohol and 12 g. of sulfuric acid of 66 Be. The mixture was refluxed for 6 hours. The mixture was extracted with chloroform, the solvent removed and the residue distilled. 49 g. of ethyl p-trifluoromethylthiophenoxy isobutyrate are obtained. (Yield: 65%, boiling at 5 mm.: 114.5 C.)

Other compounds useful in the practice of the methods of treatment of this invention are the corresponding isopropyl or n-pentyl p-trifiuoromethylthiophenoxyisobutyrate. Such corresponding compounds are produced by employing instead of 100 ml. of ethanol, ml. of anhydrous isopopyl or m1. of l-pentyl alcohol.

EXAMPLE III Ethyl p-trifluoromethylsulfinylphenoxyisobutyrate Stages A through iF are the same as those described in Example II.

Stage G, p trifluoromethylsulfinylphenoxyisobutyric acid.Into a 2 liter three-necked flask, equipped with a mechanical agitator, a thermometer and a dropping funnel, are introduced 800 ml. of methanol, a solution of 40 g. of sodium metaperiodate in 370 m1. of Water and finally 50 g. (0.178 mole) of p-trifluoromethylthiophenoxyisobutyric acid. The mixture is maintained under agitation at room temperature for 4 /2 hours. When the reaction is completed, the product, having been recovered several times in chloroform, is extracted. 44 g. of p-trifluoromethylsulfinylphenoxyisobutyric acid are obtained. (Yield: 83%, M.P. 123 C.)

Stage H, ethyl p trifiuoromethylsul-finylphenoxyisobutyrate.The esteri fication of p-trifluoromethylsulfinylphenoxyisobutyric acid is effected as described in Example II, stage G. The corresponding ester is obtained. (Yield: 58%, boiling at 5 mm.: l22124 C.).

EXAMPLE IV Ethyl p-trifluoromethylsulfonylphenoxyisobutyrate six weeks. The lithogenous regime is a normal nourishment for mice supplied by the U.A. R. Establishments, with the addition of 0.5% dosage of sodium dehydrochlorate (cf. Francois Besangon et al. 1965-1966).

The lot tested received no other treatment.

One of the medicaments of the invention was administered to each of 3 other lots subcutaneously in dosages of 36 mg., 6 days per week for 6 weeks.

.At the end of the treatment, the mice were sacrificed and autopsies performed on them. The weight of the animals, the weight and color of their livers, the dimensions of the gall bladder, the presence of macroscopically visible stones and observations by polarizing microscope were noted. The findings are given in the following table.

TABLE I Vesicle Weight Weight Percent of weight Dimenmouse liver Lithisions, Treatment in, g. in, g liver Color of liver asis mm. Birefrlngent crystals 1. 68 5. 6 Colorless 7 x3 Numerous large masses. 28 1.67 0 5 5x25 Normal. 28 1. 88 6 x3 Many large masses. 2 6.51: 3 Normal. 25 1. 04 7. 5 x3 Many large masses normal. 30 3.25 O 6 x2. 5 Norma 20 2. 60 0 4 x 1 Do. 30 2. 30 O 6 x 8 Do. 22 3. 85 0 6 x2 Small No. of large masses many small masses. 20 3. 50 0 11 x2 Normal. 24 3. 60 O 5 x l. 5 Numerous very small masses. 25 4. 80 O 6 x 2. 5 Do. 21 3. 92 0 7. 5 x 4 Normal. 20 3. 70 0 7 x 2 Do.

N0'rE.-Compound I=Ethyl p-trifluoromethylsulfonylphenoxyisobutyrate; Compound II=Ethyl p-trifiuoromethylthiophenoxyisobutyrate;

Compound 1II=Ethyl p-trifiuoromethylphenoxyisobutyrate.

hydrogen peroxide in 110 volumes. When the reaction is completed, the acetic acid is dissolved under vacuum. The crystallized product at the bottom of the flask is recovered by water, dried, washed several times with water, then dried at 50 C. 55 g. of p-trifluoromethylsulfonylphenoxyisobutyric acid are obtained. (Yield: 85%, M.-'P.: 101 C.)

Stage H, ethyl p trifluoromethylsulfonylphenoxyisobutyrate.-The esterification of p-trifluoromethylsulfonylphenoxyisobutyric acid is eiiected as illustrated in Example II, stage G. Ethyl p-trifiuoromethylsulfonylphenoxyisobutyrate is obtained. (Yield: 61%, boiling at 5 mm.: 149-l52 C.)

The compositions of this invention have been the subject of pharmacological and clinical studies to determine both their nontoxicity, their activity in controlling cholesterolemia and in preventing biliary lithiasis.

The toxicity tests of the medicaments of this invention carried out on mice weighing an average of 22 g. showed perfect tolerance of doses up to 120 mg. per mouse. ml.)

The pharmacological studies of the medicaments of the invention showed significant protection against lithiasis of the gall bladder as well as protection against cholesterolemia.

The preventive action in lithiasis of the gall bladder was studied in accordance with the test of Francois Besancon and C011. described in Socit Mdicale des Hopitaux de Paris 1966, 117, No. 2, 127-138.

Four lots of five female mice three months old, of Swiss-Git breed were tested by lithogenous regime for Table I demonstrates that in the five test mice subjected to the lithogenous regime with no other treatment, four showed macroscopically visible lithiasis and the polarizing microscope showed large cohesive doubly refractive crystals. In the fifth mouse, microscopic study showed normal bile and absence of lithiasis. The normal aspect of the bile is demonstrated by the existence of few doubly refractive crystals of small size and a maximum of 2 or 3 larger crystals in the gall bladder content.

On the other hand, in the animals treated by the lithogenous regime supplemented by the medications of this invention, no lithiasis was observed. Only 2 mice showed small crystals under microscopic examination, but in greater than normal numbers. a

The other mice were normal under microscopic examination.

Thus the significant efiicacy of the medicaments of this invention against gall bladder lithiasis is shown experimentally.

The hypochloesterol control activity of the compositions of this invention were studied according to the technique of 'I'horp and Waring (Nature 1962, 194, 948 49) as described hereafter.

The animals (male rats Wistar) received orally a combination of androsterone and the medication under study incorporated in the diet in rates of 0.01% and 0.25% respectively for 11 days.

At the conclusion of the experiment, the rats were sacrificed in parts of the carotides and the cholesterol was given in doses to each animal by the method of Bloor.

The results for three of the compositions of this invention are given in the following table.

TABLE 11 Average weight in, g. Daily intake of Cholester- No. 01 Before After Gain, nourisha, Treatment animals testing testing percent ment in, g. g./l.

5 162 226 39 87 0. 92 Androsterone. 5 187 249 33 97 0. 90 Androsterone, Compound 1-. 1 4 186 270 45 89 0. 76 Androsterone plus Compound IL. 5 151 198 31 0. 63 Androsterone plus Compound III 5 168 208 23 83 0. 61

1 One animal losing weight because of a microbial infection was eliminated.

The comparison of the cholesterolemias of the animals treated with androsterone or in combination with compounds of the invention was made by comparative analysis. The results show a highly significant hypocholesterolemia action by three of the compounds of this invention.

Tests were conducted to determine the lethal dosage in mice of compounds used in the methods of treatment of this invention. The compounds tested were:

A =Ethyl p-trifluoromethylthiophenoxyisobutyrate B=Ethyl p-trifluromethylphenoxyisobutyrate C=Ethyl p-trifluoromethylsulfonylphenoxyisobutyrate At 320 mg./kg. of body weight dosage, the death rate was:

A=0 percent B=0 percent O=0 percent The lethal dose is therefore above 320 mg./ kg.

The compounds of this invention therefore are entirely safe to use in daily dosage of to 360 mg./kg. of body weight.

The pharmacological testing shows that the methods of treatment of this invention are safe and effective in the treatment of cholesterolemia and suppress biliary lithiasis in mammals.

The pharmacological results were confirmed in human therapy, the compositions being administered in the form of capsules in dosages ranging from 1 to 2.5 grams per day.

What is claimedis:

1. The method of treating cholesterolemia in mammals to reduce the concentration of cholesterol in the blood of the mammal treated which comprises administering orally or parenterally a daily dosage of 10 to 360 mg./kg. of body weight of the mammal treated to a mammal a fluorinated derivative of the ester of phenoxy isobutyric acid of the formula:

C O O R CHQ- -CH:

in which R is a straight or branched chained alkyl having 1 through 5 carbon atoms and X is trifluoromethyl, trifiuoromethylthio, trifluoromethylsulfinyl or trifluoromethylsulfonyl.

2. The method of claim 1 in which the daily dosage is 140 to 280 mg./ kg. of body weight.

3. The method of claim 1 in which the fluorinated derivative is ethyl p-trifluoromethylphenoxyisobutyrate.

4. The method of claim 1 in which the fluorinated derivative is ethyl p-trifluoromethylthiophenoxyisobutyrate.

5. The method of claim 1 in which the fiuorinated derivative is ethyl p-trifluoromethylsulfinylphenoxyisobutyrate.

6. The method of claim 1 in which the fluorinated derivative is ethyl p-trifluoromethylsulfonylphenoxyisobutyrate.

References Cited Hansch et al., J. Am. Chem. Soc, 2817 (1963).

ALBERT T. MEYERS, Primary Examiner F. E. WADDELL, Assistant Examiner 

